CN117232928B - Milk powder matrix standard sample containing vanillin and preparation method thereof - Google Patents

Abstract

The invention discloses a milk powder matrix standard sample containing vanillin and a preparation method thereof. The invention uses electrostatic spray granulation to encapsulate the target substance, wherein vanillin is the encapsulated substance, chitosan and α -Lactalbumin is an encapsulated substance, and chitosan is the only positively charged polysaccharide in nature. The electrostatic attraction between it and α-lactalbumin may be one of the factors that promotes granulation. Electrostatic spray granulation is very good. The stability of vanillin's own chemical properties is retained. The advantage of freeze-drying is that it can maintain the integrity of various ingredients and structures in the product during the drying process, thus ensuring the integrity of the product's nutrients and active ingredients. For the standard industry Said, this is very important; through the method of combining electrostatic granulation and freeze-drying, the present invention reduces the possibility of matrix effect between vanillin and milk itself, and improves the uniformity and uniformity of vanillin in milk powder. stability.

Description

A milk powder matrix standard sample containing vanillin and its preparation method

Technical field

The invention relates to the technical field of food testing, and specifically relates to a milk powder matrix standard sample containing vanillin and a preparation method thereof.

Background technique

Vanillin, also known as vanillin, is a white or light yellow needle-shaped or crystalline powder with a melting point of 82-83°C. It is soluble in 125 times of water, 20 times of ethylene glycol and 2 times of 95% ethanol. It is soluble in chloroform. and spices. Methyl vanillin, white needle crystal (crystallized in ether), melting point (°C): 42-43°C, has a sweet woody and vanilla-like aroma, and a strong vanilla-like sweetness.

Vanillin is a substance with a strong milky aroma. It can be added to food to give it a unique milky aroma. It is widely used in the food, beverage, spice and pharmaceutical industries. Vanillin has a variety of pharmacological effects, such as antioxidant, anti-diabetic, antibacterial, anti-inflammatory, etc. However, excessive intake can also cause eating disorders, dizziness, nausea, difficulty breathing, and even damage to the liver and kidneys. Restricted by relevant regulations, The contamination of vanillin in stage 1 milk powder will not be introduced actively. However, during the production conversion process of milk powder with different phases and stages, due to incomplete equipment cleaning and workshop clearance, or the raw and auxiliary materials of the milk powder itself are not sensitive to flavor ingredients. Quality control may cause contamination migration of vanillin, which is also the cause of pollution incidents in recent years. In recent years, market regulatory authorities have repeatedly detected vanillin in Stage 1 milk powder. Therefore, it is necessary to strengthen the monitoring of vanillin content in milk powder.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a milk powder matrix standard sample containing vanillin and a preparation method thereof. The prepared milk powder matrix standard sample has undergone strict homogeneous stability testing and has the preparation method The method is simple and effective, has good uniformity and high stability.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A method for preparing a milk powder matrix standard sample containing vanillin, including the following steps:

S1. Stir the milk powder and water evenly to obtain the reconstituted emulsion;

S2. Pour the reconstituted emulsion into the freeze-drying tray and perform freeze-drying;

S3. Mix chitosan, α-lactalbumin and vanillin evenly to obtain a mixture, then add the mixture to the methanol aqueous solution and mix evenly to obtain the target solution;

S4. Stir the target solution first, then connect it to the electrostatic spray device, set the injection rate, adjust the voltage and the distance of the receiving plate, perform electrostatic granulation, and use the freeze-drying plate that has been freeze-dried in step S2 to receive the particles;

S5. After the particles are received, continue to pour the recovery emulsion into them and perform the freeze-drying operation;

S6. Move the freeze-dried sample in step S5 into a grinder, grind it through a 30-mesh sieve, and mix the residue evenly with a high-efficiency mixer;

S7. Vacuum-pack the sample obtained in step S6 and store it in a refrigerator to obtain the milk powder matrix standard sample.

Preferably, in step S1, the mass ratio of milk powder and water is 1:10-15.

Preferably, in step S2, the thickness of the reconstituted emulsion poured into the freeze-drying tray is 1-2cm.

Preferably, in step S2, the specific steps of the freeze-drying process are as follows: pre-freezing at -80°C for 3 hours, freeze-drying at -40°C for 3 hours, freeze-drying at -30°C for 3 hours, freeze-drying at -20°C for 3 hours, and freeze-drying at -10°C for 8 hours. , freeze drying at 0°C for 8 hours, freeze drying at 10°C for 6 hours, and freeze drying at 20°C for 8 hours.

Preferably, in step S3, the mass ratio of chitosan, α-lactalbumin and vanillin is 1:2:1.

Preferably, in step S3, the mass ratio of the mixture and the methanol aqueous solution is 3-4:100, and the mass fraction of the methanol aqueous solution is 5%.

Preferably, in step S4, the sampling rate is 3.0-3.5mL/h, the voltage is 20V, and the receiving distance is 20cm.

Preferably, in step S5, the thickness of the restored emulsion is 1-2cm.

Preferably, in step S5, the freeze-drying operation steps are as follows: pre-freezing at -80°C for 3 hours, freeze-drying at -40°C for 3 hours, freeze-drying at -30°C for 3 hours, freeze-drying at -20°C for 3 hours, freeze-drying at -10°C for 8 hours, 0 The treatment was carried out in the order of freeze-drying at 10°C for 8 hours, freeze-drying at 10°C for 6 hours, and freeze-drying at 20°C for 8 hours.

The present invention also provides a vanillin-containing milk powder matrix standard sample prepared by the above preparation method.

Compared with the prior art, the present invention has the following beneficial effects:

(1) Since vanillin is easily oxidized slowly in humid air, and considering the complexity of the milk powder matrix, the present invention uses electrostatic spray granulation to encapsulate the target substance, in which vanillin is the encapsulated substance and chitosan is the encapsulated substance. Sugar and α-lactalbumin are encapsulated substances. Chitosan is the only positively charged polysaccharide in nature. The electrostatic attraction between it and α-lactalbumin may be one of the factors that promotes granulation. Electrostatic spray granulation The stability of vanillin's own chemical properties is well retained. The advantage of freeze-drying is that it can maintain the integrity of various ingredients and structures in the product during the drying process, thus ensuring the integrity of the nutritional ingredients and active ingredients of the product. For For the standard industry, this is very important; through the method of combining electrostatic granulation and freeze-drying, the present invention reduces the possibility of matrix effect between vanillin and milk itself, and improves the concentration of vanillin in milk powder. Uniformity and stability.

(2) The matrix standard sample prepared by the present invention has undergone strict homogeneous stability testing and has the advantages of good uniformity and high stability. It has been proved by inspection that this standard can ensure product quality whether it is transported under frozen or high temperature conditions, meets the transportation requirements of different regions, has a long shelf life, and is an effective and reliable physical standard sample that can be used in related detection and analysis fields. Quality control can also be used as a means to certify and assess the analytical capabilities of laboratories or technicians participating in testing, which has significant economic value and market competitiveness.

(3) The vanillin-containing milk powder matrix standard sample prepared by the present invention is mainly used in laboratory proficiency verification, internal quality control, method verification and other activities; it is helpful for laboratory quality control and ensures the accuracy of quantitative detection results. ; It helps to strengthen the quality and safety control of milk powder production and improve the testing level of inspection and testing institutions.

(4) Uniformity is a basic attribute of standard materials. Due to the difficulty of preparation methods, high investment, long investment time, unclear metabolism of target components and other factors, it is necessary to obtain qualified uniformity of real physical standard samples. It is difficult to prepare standard samples, but the present invention determines that under specified conditions, the prepared samples can obtain better uniformity.

(5) Stability is also a basic attribute of standard materials. Standard samples involve storage and transportation processes from preparation to use, which may lead to changes in the stability of standard samples. Therefore, two aspects of stability assessment are required. One is based on sample packaging. and transportation form, select the temperature for short-term stability evaluation, usually conducted under different temperature conditions, to examine the impact of temperature on the characteristic values of the standard material; second, under the specified storage conditions, regularly test the standard over a longer period The material characteristic values were tested to examine its ability to remain within the specified range. The short-term stability and long-term stability of the standard sample prepared according to the present invention met the requirements, and its validity period was 6 months in a -18°C storage environment.

Description of drawings

Figure 1 is a mass spectrum of the milk powder matrix standard sample prepared in Example 1 of the present invention.

Detailed ways

The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following examples.

It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

Example 1

A method for preparing a milk powder matrix standard sample containing vanillin, including the following steps:

S1. Stir pure milk powder (Mengniu) and first-grade water at a mass ratio of 1:10 and mix evenly. The speed of the overhead stirrer is 500r/min and the stirring time is 20min to obtain the reconstituted emulsion;

S2. Pour the restored emulsion into the freeze-drying tray with a thickness of 1cm. Pre-freeze at -80℃ for 3h, freeze-dry at -40℃ for 3h, freeze-dry at -30℃ for 3h, freeze-dry at -20℃ for 3h, and freeze at -10℃. The emulsion was freeze-dried in the order of drying for 8 hours, freeze-drying at 0°C for 8 hours, freeze-drying at 10°C for 6 hours, and freeze-drying at 20°C for 8 hours;

S3. Mix chitosan, α-lactalbumin and vanillin in a mass ratio of 1:2:1 to obtain a mixture. Then add 3g of the mixture into 100g, 5wt% methanol aqueous solution and mix evenly to obtain the target solution;

S4. Stir the target solution with an overhead stirrer at 400r/min for 30 minutes. Connect the target solution to the electrostatic spray device. Set the injection rate to 3.0mL/h, the voltage to 20V, and the receiving distance to 20cm to perform electrostatic spraying. Granules, use the freeze-drying tray that has been freeze-dried in step S2 to receive the granules;

S5. After the particles are received, continue to pour the recovery emulsion into them. The thickness of the recovery emulsion is 1cm. Pre-freeze at -80℃ for 3h, freeze-dry at -40℃ for 3h, freeze-dry at -30℃ for 3h, and -20℃. The emulsion was freeze-dried in the order of 3 hours of freeze-drying, -10°C freeze-drying for 8 hours, 0°C freeze-drying for 8 hours, 10°C freeze-drying for 6 hours, and 20°C freeze-drying for 8 hours;

S6. Move the freeze-dried sample in step S5 into a knife grinder, crush it for 50 minutes at 2000r/min, pass it through a 30-mesh sieve, and mix the material under the sieve with a high-efficiency mixer at 100r/min for 60 minutes;

S7. Vacuum-pack the sample obtained in step S6 and store it in a refrigerator at -18°C to obtain a milk powder matrix standard sample containing vanillin.

Comparative example 1

What is different from Example 1 is that in step S3, chitosan and vanillin are mixed according to a mass ratio of 1:1.

Comparative example 2

What is different from Example 1 is that in step S3, α-lactalbumin and vanillin are mixed according to a mass ratio of 2:1.

Comparative example 3

Different from Example 1, in step S3, the mass ratio of chitosan, α-lactalbumin and vanillin is 2:2:1.

Comparative example 4

Different from Example 1, in step S3, the mass ratio of chitosan, α-lactalbumin and vanillin is 1:2:2.

The milk powder matrix standard sample was tested in accordance with GB 5009.284-2021 "National Food Safety Standard for Determination of Vanillin, Methyl Vanillin, Ethyl Vanillin and Coumarin in Foods". The test results are shown in Figure 1 , it can be seen from the detection spectrum of the milk powder matrix standard sample that the vanillin in the standard sample prepared in Example 1 of the present invention has a good separation effect.

Uniformity test

The samples prepared in Example 1 and Comparative Examples 1-4 were tested for uniformity. The detection method adopted GB5009.284-2021 "National Food Safety Standard for Vanillin, Methyl Vanillin, Ethyl Vanillin and Vanillin in Foods". "Determination of legumin", the statistical method adopts the single-factor variance statistical analysis method, and uses the F test to conduct a uniformity test on the three levels of samples. The specific method is to randomly select 30 bottles from the samples prepared in each group above, and each sample is as Independent sub-samples are tested, and each sub-sample also needs to be measured three times in parallel. The experimental results are the average of the parallel measurements. All samples are tested in random order under repeatability conditions. The test results are as follows:

Table 1 Example 1 Sample Uniformity Evaluation Measurement Result Recording Table

Table 2 Comparative Example 1 Sample Uniformity Evaluation Measurement Result Recording Table

Table 3 Comparative Example 2 Sample Uniformity Evaluation Measurement Result Recording Table

Table 4 Comparative Example 3 Sample Uniformity Evaluation Measurement Result Recording Table

Table 5 Comparative Example 4 Sample Uniformity Evaluation Measurement Result Record Table

Performing single-factor variance analysis on the above data, the following variance analysis results can be obtained: when the confidence probability is 0.95, the degrees of freedom f1=29, f2=60, it can be concluded through the F test table that F0.05 (29, 60 ) = 1.65. The F values of the test results of the two sets of samples of Comparative Example 1 and Comparative Example 2 are both higher than F0.05 (29, 60) = 1.65, indicating that the uniformity of Comparative Examples 1-2 does not meet the requirements of the standard sample. Therefore, it has no value as a standard sample; the F value of the two groups of samples of Comparative Example 3 and Comparative Example 4 is < the critical value F0.05 (29, 60), indicating that there is no significant difference within and between the two groups of samples. The samples are uniform and meet the requirements as quality control samples. However, the F values of the two groups of samples are greater than those of Example 1, indicating that the uniformity of Comparative Examples 3 and 4 is not as good as that of Example 1. At the same time, the precision RSD also reflects that of Example 1. The sample is worse than the two groups of Comparative Example 3 and Comparative Example 4, indicating that the electrostatic spray material and ratio have a significant impact on the uniformity of the matrix standard sample finally prepared by this method.

Stability test

The stability of the samples meeting the uniformity requirements was tested, and the samples prepared in Example 1 were selected for experiments. According to "JJF 1343-2022 Value Setting, Uniformity and Stability Evaluation of Standard Materials", the statistical method for stability testing is determined to be the t analysis test method, and the characteristic value change curve with time is used to determine whether the sample characteristic value has a unidirectional change trend. , use a linear fitting model to evaluate the stability of standard samples. Stability testing includes two aspects: long-term stability (storage stability) and short-term stability (transportation stability). Sampling is carried out according to the sampling principle of dense first and then thin. Among them, a total of 6 sampling time points are set for short-term stability, 4 ℃, 50 ℃ storage, long-term stability testing set up a total of 6 sampling time points, -18 ℃ storage, each sampling time point randomly selected 3 samples in Example 1, each sample was done twice in parallel, and three sub-samples were taken. The vanillin content in the sample of Example 1 was analyzed using the average value of the samples. The test results are as follows:

Table 6 Short-term stability results of vanillin in the sample of Example 1 at 4°C

Table 7 Short-term stability results of vanillin in the sample of Example 1 at 50°C

Table 8 Long-term stability results of vanillin in the sample of Example 1

It can be seen from Table 6 to Table 8 that |b1| of the sample prepared by the method of Example 1 of the present invention is less than t0.95 in both the short-term stability and the long-term stability of six months, n-2×s(b1 ), the vanillin content in the matrix standard sample tends to be stable, and the storage environment of the matrix standard sample can be determined at -18°C, and the validity period is 6 months.

Value

The value of the sample prepared in Example 1 was determined according to the requirements of "JJF 1343-2022 Valuation and Uniformity and Stability Evaluation of Standard Materials". The value of the matrix standard sample was determined using the collaborative value determination method of 8 laboratories, and the laboratories participating in the determination were all accredited laboratories. The test results need to check whether the data obeys the normal distribution. Secondly, Grubbs is used to test whether the data in each laboratory has suspicious values, and the Cochrane test is used to determine whether the data between laboratories have the same accuracy. When the above requirements are met, Only under certain conditions can the data be representative and can participate in the statistical analysis of the fixed value results. The results are shown in the following table:

Table 9 Inter-laboratory measurement data of standard samples

The normality test uses the Shapiro-Wilk test. Looking up the table, we can get W(n,p)=0.842 (where n=10, p=0.95). Since the W values of each laboratory data are greater than 0.842, so it can be considered that the test data from each laboratory are accepted as a normal distribution.

The Grubbs test method was used to test whether the test results within each laboratory group have suspicious values. From the Grubbs critical value table, it can be seen that λ _(0.05,10) =2.290. The results show that the absolute value of the maximum residual value of the test results is less than λ _{(0.05 ,10)} *S, * represents the multiplication sign ×, indicating that there are no abnormal values and no suspicious values between the fixed value results of each laboratory, so all data are retained and can participate in the statistics of fixed value results.

The Cochrane test is used to determine whether the value results of each laboratory are of equal accuracy. The Cochrane test requires that C be less than or equal to C _(α,m,n) , indicating that the average values of each group of data are of equal accuracy. Otherwise, it will be judged as an outlier, and this group of data should be removed when calculating the fixed value result. Looking at the critical table, we can see that C _(0.05,8,10) =0.2829. Table 9 shows that the C value of the Cochrane test is 0.2184, which is less than the critical value. The data between laboratory test results are of equal accuracy. All data should be retained and can participate in fixed value result statistics. The average test results of 8 laboratories See Table 10.

Table 10 Average test results of 8 laboratories

After conducting the suspicious value test and equal accuracy test on the value results of each laboratory, it is also necessary to test whether there is a significant difference in the average value of the value results of each laboratory and whether the normality of the data distribution is met. After calculation, there is no significant difference between the average values of the eight laboratories and they meet the normality test. The data meets the statistical requirements. Then the standard value of the vanillin-containing milk powder matrix standard sample prepared in Example 1 is taken from each laboratory. The average value of the laboratory average results, that is, the standard value of the vanillin-containing milk powder matrix standard sample prepared in Example 1 is 1.36 mg/kg.

uncertainty

According to "JJF 1343-2022 Valuation, Uniformity, and Stability Evaluation of Standard Materials", the uncertainty of the value determination results of the standard material consists of three parts: the uncertainty U _bb caused by the non-uniformity of the standard material, the uncertainty of the standard material The uncertainty U _sts caused by instability and the uncertainty U _char caused by the standard material determination process. The synthetic uncertainty U(y) can be determined by calculating the uncertainty of each part, with a confidence level of 95%, and the expanded uncertainty U is twice the synthetic uncertainty:

U = 2×U _(y)

(1) Uncertainty introduced by uniformity

According to "JJF 1343-2022 Valuation, Uniformity and Stability Evaluation of Standard Materials", the within-bottle variance of the sample of Example 1 is smaller than the between-bottle variance of the sample ( ), so it can be estimated that the standard deviation of the uniformity between bottles s _bb is equal to the uncertainty component U _bb caused by the inhomogeneity between bottles, so U _bb =0.0305 mg/kg.

(2) Uncertainty introduced by stability

According to the requirements of "JJF 1343-2022 Valuation, Uniformity and Stability Evaluation of Standard Materials", when the trend analysis method is used for stability testing, the stability change trend of the sample in Example 1 is not obvious. The uncertainty introduced by stability can be calculated according to the formula: U _sts =s(β ₁ )·X. It is known that s(β ₁ )=0.00341 and X=6 months for this standard material stability monitoring, so U _sts =0.0205 mg/kg.

(3) Uncertainty introduced by fixed value

The method used to determine the sample value in Example 1 is to use a variety of methods with confirmed accuracy, and to cooperate with multiple laboratories to determine the value. Each laboratory only provides a series of observation values. According to the standard requirements, in principle, this The standard deviation of the average value in a fixed value mode is the fixed value uncertainty U _char .

The s value is the standard deviation of the mean value of the determination results of eight laboratories. The p value is the total number of participating laboratories, which is 8. After calculation, the standard uncertainty introduced by the joint laboratory determination is finally obtained: U _char =0.0025 mg /kg.

Therefore, the total synthesis uncertainty U _(y) can be calculated to be 0.037 mg/kg, and the expanded uncertainty U is 0.074 mg/kg. The final determined characteristics of vanillin in the milk powder matrix standard sample of Example 1 The value is 1.36±0.074 mg/kg (k=2).

Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention shall fall within the scope of protection claimed by the present invention.

Claims (10)

1. The preparation method of the standard sample of the milk powder matrix containing vanillin is characterized by comprising the following steps of:

s1, uniformly stirring and mixing milk powder and water to obtain a reconstituted emulsion;

s2, pouring the reconstituted emulsion into a freeze-drying disc for freeze-drying treatment;

s3, uniformly mixing chitosan, alpha-lactalbumin and vanillin to obtain a mixture, adding the mixture into a methanol aqueous solution, and uniformly mixing to obtain a target solution;

s4, stirring the target solution, then, connecting the target solution into an electrostatic spraying device, setting a sample injection rate, adjusting voltage and a receiving disc distance, performing electrostatic granulation, and receiving particles by adopting a freeze-drying disc subjected to freeze-drying treatment in the step S2;

s5, after the particles are received, continuously pouring the reconstituted emulsion into the particles, and performing freeze-drying operation;

s6, transferring the freeze-dried sample obtained in the step S5 into a pulverizer, pulverizing, sieving with a 30-mesh sieve, and uniformly mixing the undersize materials by using a high-efficiency mixer;

and S7, vacuum packaging the sample obtained in the step S6, and refrigerating and preserving to obtain the milk powder matrix standard sample.

2. The method for preparing a standard sample of milk powder base containing vanillin according to claim 1, wherein in step S1, the mass ratio of milk powder to water is 1:10-15.

3. The method for preparing a standard sample of powdered milk matrix containing vanillin according to claim 1, wherein in step S2, the reconstituted emulsion is poured into a lyophilization tray to a thickness of 1-2cm.

4. The method for preparing a standard sample of milk powder base containing vanillin according to claim 1, wherein in step S2, the specific steps of the lyophilization process are as follows: the treatment is carried out according to the sequence of pre-freezing at-80 ℃ for 3h, freeze-drying at-40 ℃ for 3h, freeze-drying at-30 ℃ for 3h, freeze-drying at-20 ℃ for 3h, freeze-drying at-10 ℃ for 8h, freeze-drying at 0 ℃ for 8h, freeze-drying at 10 ℃ for 6h and freeze-drying at 20 ℃ for 8 h.

5. The method for preparing a standard sample of milk powder base containing vanillin according to claim 1, wherein in step S3, the mass ratio of chitosan, α -lactalbumin and vanillin is 1:2:1.

6. The method for preparing a standard sample of milk powder base containing vanillin according to claim 1, wherein in step S3, the mass ratio of the mixture to the aqueous methanol solution is 3-4:100, and the mass fraction of the aqueous methanol solution is 5%.

7. The method for preparing a standard sample of powdered milk matrix containing vanillin according to claim 1, characterized in that in step S4, the sample injection rate is 3.0-3.5mL/h, the voltage is 20V, and the receiving distance is 20cm.

8. The method for preparing a standard sample of powdered milk base containing vanillin according to claim 1, wherein in step S5, the pouring thickness of the reconstituted emulsion is 1-2cm.

9. The method for preparing a standard sample of milk powder base containing vanillin according to claim 1, wherein in step S5, the lyophilization operation is as follows: the treatment is carried out according to the sequence of pre-freezing at-80 ℃ for 3h, freeze-drying at-40 ℃ for 3h, freeze-drying at-30 ℃ for 3h, freeze-drying at-20 ℃ for 3h, freeze-drying at-10 ℃ for 8h, freeze-drying at 0 ℃ for 8h, freeze-drying at 10 ℃ for 6h and freeze-drying at 20 ℃ for 8 h.

10. A standard sample of a powdered milk matrix containing vanillin prepared by the method of any of claims 1-9.